Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls.
Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-[5-O-(trans-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-O-be ta-D- xylopyranosyl-(1----4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 1H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. Vmax was determined to be 1.17 mumol min-1 mg-1 and Km 13.2 microM at pH 7.5 and 30 degrees C.
Borneman
W S
WS
Richard B. Russell Agricultural Research Center, U.S. Department of Agriculture, Athens, Georgia 30613.
Hartley
R D
RD
Himmelsbach
D S
DS
Ljungdahl
L G
LG
D002240:Carbohydrate Sequence; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D003470:Culture Media; D005656:Fungal Proteins / Q000378:metabolism; D005658:Fungi / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010944:Plants / Q000378:metabolism*; D006109:Poaceae / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D014312:Trisaccharides / Q000378:metabolism*
D003470:Culture Media; D005656:Fungal Proteins; D014312:Trisaccharides; C066379:O-(5-O-(4-coumaroyl)-alpha-arabinofuranosyl)-(1-3)-O-beta-xylopyranosyl-(1-4)-xylopyranose; D002265:Carboxylic Ester Hydrolases; C067271:trans-4-coumaroyl esterase